| Background Forensic evidence materials, are often subjected to harsh environments that degrade the DNA. Moreover, some samples (e.g. hairs, teeth or bone) often contain low amounts of DNA,which are hard to type. When nuclear DNA cannot be obtained, sequencing of mitochondrial DNA (mtDNA) is a commonly used alternative for these samples. There is a higher DNA copy number of mtDNA per cell than nuclear DNA. In addition, the mitochondrial genome has a 10 times higher nucleotide mutation rate than the nuclear genome. Especially the two hypervariable regions HVI and HVII, located in the non-coding control region, are dense in polymorphic positions. The extensive sequence variability found within the control region makes this region suitable for routine analysis in human identification. However, due to maternal inheritance and the resulting lack of recombination of this genome, there is a relatively high risk of mtDNA sequence identity by chance. Although a majority (84%) of the HVI/HVII mtDNA sequences is found to be unique in a large sample set, several individuals share identical sequences. The significance of identification test is to deplete identity, utilizing mtDNA polymorphism. Mitochondrial DNA is characterized by clusters of closely related lineages, or haplogroups, which are distinguished by certain diagnostic polymorphisms, in population genetics. The diagnostic SNPs that define these haplogroups are commonly located in the coding region, and some are more common in certain populations. Besides the polymorphism-dense non-coding region, a substantial number of polymorphic positions are found in the 15,447 bp coding region, but with a lower average variability. Therefore, analysis of positions in the coding region has been suggested as a valuable tool to increase the discrimination power in forensic mtDNA analysis.Objective To establish a simple, fast and economical protocol based on the ripe technology of SNaPshot (minisequencing), and survey the frequency distribution of 24 SNPs of mtDNA code region in Han population of Wuhan, then provide a basis for forensic application.Methods To select 24 mtDNA-SNP loci with suitable polymorphism, ascertain their base sequence and design the target fragment primers and single base extension primers by correlated software. To establish a fluorescent-multiplex PCR system of 24 mtDNA-SNP loci by firstly amplifying the allele fragment of diverse locus using special allele PCR technology, secondly processing the SBE reaction with the purified PCR product, and finally identifying the base type with fluorescent assay.Results A fluorescent-multiplex PCR system of 24 mtDNA-SNP loci was established. The typing results showed that each group had eight peaks with diverse color, and the fragment sizes of alleles among different SNP loci were different. Even the different base of a SNP locus located at a variant position. In 100 samples, there are 30 haplotypes, while the frequency range is 0.01~0.04. The haplotype diversity of these 24 loci was estimated to be 0.956 in Wuhan Han population. Conclusion The SNP multiplex amplification system set in this study is simple and economical, and is of a high application value in forensic medicine.
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